Synthetic oligonucleotides, such as antisense molecules, aptamers, ribozymes and RNA interference (RNAi) molecules, are increasingly used in biomedical research, diagnostics and therapeutics. These synthetic oligonucleotides have been used to inhibit or knock-down expression of a gene in vitro, in situ, and in vivo in a sequence dependent manner.
It is frequently useful to attach or link targeting ligands or other pharmacological modifiers to synthetic oligonucleotides, especially for therapeutic in vivo delivery. To be useful, the linkage chemistry should be modular, so that it is readily adaptable to different synthetic oligonucleotides as well as different targeting ligands and pharmacological modifiers. In addition, the linkage chemistry should have simple reaction conditions, be efficient (i.e. give high chemical yields), not require toxic or other detrimental products, and not produce toxic or other detrimental byproducts. The linkage chemistry should also be stable outside of the target cell, such as in circulation, subcutaneous space, or extracellular space, but be readily cleavable at the final site of action, such as inside the target cell.
An example of such a reaction useful in linking synthetic oligonucleotides to targeting ligands or other pharmacological modifiers is the cycloaddition of cyclic alkynes and azides, which is one of the reactions known as “click reactions” or “click chemistry”. In click reactions, two separate molecular entities, one charged with an azide and one charged with a strained cycloalkyne, will spontaneously combine into a single molecule by a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC). The reaction is mild in nature, rapid, and high-yielding and occurs at about physiological pH, in water, and in the vicinity of biomolecular functionalities. This reaction has become a versatile tool for bioorthogonal labeling and imaging of biomolecules (e.g. proteins, lipids, glycans and the like), proteomics and materials science. The cycloaddition reaction proceeds spontaneously, in the absence of a catalyst. Metal-free cycloadditions are also referred to as “metal-free click reactions”. The power of SPAAC for bioorthogonal labeling lies in the fact that an isolated cyclic alkyne or azide is fully inert to biological functionalities, such as for example amines, thiols, acids or carbonyls, but in combination undergoes rapid and irreversible cycloaddition, leading to a stable triazole conjugate. For example, azido-modified proteins, obtained by expression in auxotrophic bacteria, genetic engineering or chemical conversion, can be cleanly labeled with biotin, fluorophores, PEG-chains or other functionalities upon simply stirring the azido-protein with a cyclooctyne conjugate. Moreover, the small size of azide has proven highly useful for application of SPAAC in the imaging of specific biomolecules by means of the chemical reporter strategy.
We have found the alkyne disulfides currently available, such as Dibenzocyclooctyne (DBCO)—S—S—NHS,
offer insufficient stability during modification of compounds leading to poor synthetic yields and the presence of undesired impurities. We now describe improved alkyne disulfides having improved stability properties resulting in improved yields and purity.